Early Introduction of Plant Polysaccharides Drives the Establishment of Rabbit Gut Bacterial Ecosystems and the Acquisition of Microbial Functions

ABSTRACT In mammals, the introduction of solid food is pivotal for the establishment of the gut microbiota. However, the effects of the first food consumed on long-term microbiota trajectory and host response are still largely unknown. This study aimed to investigate the influences of (i) the timing of first solid food ingestion and (ii) the consumption of plant polysaccharides on bacterial community dynamics and host physiology using a rabbit model. To modulate the first exposure to solid nutrients, solid food was provided to suckling rabbits from two different time points (3 or 15 days of age). In parallel, food type was modulated with the provision of diets differing in carbohydrate content throughout life: the food either was formulated with a high proportion of rapidly fermentable fibers (RFF) or was starch-enriched. We found that access to solid food as of 3 days of age accelerated the gut microbiota maturation. Our data revealed differential effects according to the digestive segment: precocious solid food ingestion influenced to a greater extent the development of bacterial communities of the appendix vermiformis, whereas life course polysaccharides ingestion had marked effects on the cecal microbiota. Greater ingestion of RFF was assumed to promote pectin degradation as revealed by metabolomics analysis. However, transcriptomic and phenotypic host responses remained moderately affected by experimental treatments, suggesting little outcomes of the observed microbiome modulations on healthy subjects. In conclusion, our work highlighted the timing of solid food introduction and plant polysaccharides ingestion as two different tools to modulate microbiota implantation and functionality. IMPORTANCE Our study was designed to gain a better understanding of how different feeding patterns affect the dynamics of gut microbiomes and microbe–host interactions. This research showed that the timing of solid food introduction is a key component of the gut microbiota shaping in early developmental stages, though with lower impact on settled gut microbiota profiles in older individuals. This study also provided in-depth analysis of dietary polysaccharide effects on intestinal microbiota. The type of plant polysaccharides reaching the gut through the lifetime was described as an important modulator of the cecal microbiome and its activity. These findings will contribute to better define the interventions that can be employed for modulating the ecological succession of young mammal gut microbiota.


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The authors considered all reviewer comments carefully which results in a successful improvement of the manuscript. The study still included a functional prediction analyses and the authors applied the suggestion of the reviewer to use the NSTI score that's good. But this results in even less outcome. Only 1/4 of the OTUs could now be used for the prediction and the lack of information is seen by not even showing the output, Suppl Fig 1 doesn't count for me in fact. I suggest to remove the prediction from the manuscript as it doesn't give any add value to the story. Line 307 Bacteroidetes change to Bacteroidota Reviewer #3 (Comments for the Author): I reviewed an earlier version of this paper and the authors have significantly improved the work since the previous version. I have several minor comments, which should be addressed before the article is ready for publication.
References: All bioinformatic tools and datasets used in this study should be referenced e.g. VSEARCH, SWARM, SILVA.
All figures: Please include the unit of measurement for "Age" in the axis label and legends for all figures as you have in Figure 2 i.e., replace "Age" with "Age (days)".
Ln 297: "significant increase" Is this statistically significant? I cannot see any statistical values in the text or the figure. Please check if these proportion changes are significant and include the information in both the text and Figure 3.
Ln 297-298: The authors present both phylum level and family level analysis of the caecum and appendix communities in Figure  3, but the family level analysis is not even mentioned in the text. In fact, this 4-panel main figure is described in one sentence in the text. And there does appear to be some interesting findings from my initial observation of the data. For example, it appears that the increase gradual increase in Firmicutes was mostly driven by an early increase in Ruminococcaceae and then a later increase in Eubacteriaceae -although this prediction would need to be backed up with data analysis. PiCrust analysis: The authors have done a good job in addressing my PiCrust comment and mitigating the limitations of using PiCrust on less extensively sampled microbiomes. The compromise that they have chosen between accuracy and data loss seems sensible.
Ln 457-461: Please provide this data in a Supp table i.e., 48 pathways, fold change and appropriate statistics (in this case I think you need to account for multiple testing). Also please provide the statistical significance of these differences in the main text.

Manuscript mSystems00243-22
Response to the editor Dear Dr. Cotter, Thank you for giving us the opportunity to submit a revised draft of the manuscript "Early introduction of plant polysaccharides drives the establishment of rabbit gut bacterial ecosystems and the acquisition of microbial functions" for publication in the mSystems journal. We appreciate the time and effort that you and the reviewers dedicated to improve our manuscript.
We have incorporated most of the suggestions made by the reviewers. Those changes are highlighted within the manuscript. Please see below, in blue, for a point-by-point response to the reviewers' comments and concerns. All page numbers refer to the revised manuscript file with tracked changes in yellow.

Reviewer #1 (Comments for the Author):
The authors considered all reviewer comments carefully which results in a successful improvement of the manuscript.
Thank you very much for your thoughtful comments and efforts towards improving our manuscript.
The study still included a functional prediction analyses and the authors applied the suggestion of the reviewer to use the NSTI score that's good. But this results in even less outcome. Only 1/4 of the OTUs could now be used for the prediction and the lack of information is seen by not even showing the output, Suppl Fig 1 doesn't count for me in fact. I suggest to remove the prediction from the manuscript as it doesn't give any add value to the story.
Thank you for noticing this improvement. We agree with the reviewer that the reduced coverage prediction is unfortunate and should be considered with caution. This limitation was thus mentioned line 482 and better specified lines 378-379.
Although limited, we would like to save these results because (i) they contribute to provide a global assessment of the bacterial ecosystems (335 pathways evaluated from 72% of the total relative abundances), (ii) the accuracy of the remained predictions was proven and allows us a reasonable discussion.
The output of the predictions was shortly described lines 378-380. It was more developed lines 390-395 as recommended. It was also discussed lines 482-484. We believe it provided some insights into bacterial functions together with metabolomics methodology.
A supplemental table with all the pathways affected by the type of dietary polysaccharides was added to provide more outcome (Supplemental Table S4C). No specific figure was presented within the manuscript because it appears non-relevant to us to emphasize further these results.
We further believe that the supplemental figure S1 proposed can provide interesting references for people wishing to follow-up complex bacterial communities, even though this was not in the main scope of our study. We thus would like to maintain it.

Line 307 Bacteroidetes change to Bacteroidota
We do not understand this point since it appears to us that the correct taxonomic name was used. To be clearer, a mention to the taxonomic level considered was added line 318.

Reviewer #3 (Comments for the Author):
I reviewed an earlier version of this paper and the authors have significantly improved the work since the previous version. I have several minor comments, which should be addressed before the article is ready for publication.
We thank the referee for the careful and insightful review of our manuscript.
All figures: Please include the unit of measurement for "Age" in the axis label and legends for all figures as you have in Figure 2 i.e., replace "Age" with "Age (days)".
Ln 297: "significant increase" Is this statistically significant? I cannot see any statistical values in the text or the figure. Please check if these proportion changes are significant and include the information in both the text and Figure 3.
Indeed, our analysis were mainly performed within groups with no emphasize on the age effect to focus on the main scope of the study. To support these conclusions, extra statistical tests were done and are presented lines 298-301. This result was not added within figure 3 because it is not adapted to evidence the numerous age-related changes of bacterial distribution.
Ln 297-298: The authors present both phylum level and family level analysis of the caecum and appendix communities in Figure 3, but the family level analysis is not even mentioned in the text. In fact, this 4-panel main figure is described in one sentence in the text. And there does appear to be some interesting findings from my initial observation of the data. For example, it appears that the increase gradual increase in Firmicutes was mostly driven by an early increase in Ruminococcaceae and then a later increase in Eubacteriaceae -although this prediction would need to be backed up with data analysis.
We agree with the reviewer that this figure could be more extensively described. However, to limit the size of our result sections, we prefer to limit this investigation. We only added the analysis of the longitudinal trends regarding Lachnospiraceae, Ruminococcaceae and Bacteroidaceae to shed light on the next result sections since these families are mentioned. These modifications appear from lines 302 to 304. For more detailed analysis, readers can access to the references quoted [23,42]. This was added in the figure 3 and corresponding caption.
PiCrust analysis: The authors have done a good job in addressing my PiCrust comment and mitigating the limitations of using PiCrust on less extensively sampled microbiomes. The compromise that they have chosen between accuracy and data loss seems sensible.
Thank you for your feedback on this aspect.
Ln 457-461: Please provide this data in a Supp table i.e., 48 pathways, fold change and appropriate statistics (in this case I think you need to account for multiple testing). Also please provide the statistical significance of these differences in the main text. We added this detailed information in the Supplemental Table S4C. This allowed us to capture interesting modulations lines 394-395 and these points were more discussed lines 482 to 484. Significant levels were mentioned in the manuscript lines 392 and 395. Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. For your reference, ASM Journals' address is given below. Before it can be scheduled for publication, your manuscript will be checked by the mSystems production staff to make sure that all elements meet the technical requirements for publication. They will contact you if anything needs to be revised before copyediting and production can begin. Otherwise, you will be notified when your proofs are ready to be viewed.
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